For a long time, scientists have focused only on the pathological and/or histological study of biological samples. A preservation and/or stabilization of the samples for such studies was usually carried out, if at all, by placing the samples into formaldehyde solutions and/or by embedding the samples in paraffin. But even the cooling or freezing of biological samples for preservation purposes has long been current practice.
Only when it was recognized that the detection of certain constituents of biological samples such as, for example, nucleic acids or proteins, is of great benefit, in particular in the field of medical and clinical diagnostics, did it become clear that novel, more effective and more economical preservation and/or stabilization reagents and/or methods were required.
In the course of these developments, one recognized that it is precisely the status (gene expression or protein pattern) of the fresh sample constituents which are important for molecular-biological study which may undergo rapid changes, even directly after the sample has been taken from its natural environment, so that even prolonged storage of the samples in the untreated state, for example as the result of unexpected delays during transport into the laboratory or the like, may falsify a molecular-biological analysis or indeed make the latter entirely impossible.
It is precisely the nucleic acid status of a biological sample which undergoes more rapid changes as more time elapses between sampling and the analysis of the sample. The ribonucleic acids (RNAs) in particular are degraded very rapidly as the result of ubiquitous RNases. Also, the degradation of nucleic acids is accompanied by the induction of, for example, stress genes and thus the synthesis of novel mRNA molecules which likewise greatly modify the transcription pattern of the sample. It is therefore necessary immediately to stabilize the sample in order to retain the gene expression profile to be analyzed.
An immediate stabilization of the sample is necessary not only for analyzing nucleic acids, but also for detailed proteomic studies of a biological sample since the protein pattern too undergoes changes immediately after sampling. This is the result firstly of degradation or de novo synthesis, but also changes in the protein modification, such as, for example, phosphorylation/dephosphorylation, which happens very rapidly.
Since protein-chemical and molecular-biological analyses are employed not only in the field of medical and clinical diagnostics, but also increasingly in other fields such as forensics, pharmacy, food analytics, agriculture, environmental analytics and in many research projects, retaining the integrity of the molecular structure of the samples, and, in this context, their immediate stabilization, is thus a prerequisite of utmost importance in all these fields.
Over the years, a multiplicity of very different stabilizing reagents and/or methods have been developed in order to stabilize a wide range of very different biological samples.
As already mentioned at the outset, it has long been known to stabilize samples by means of aqueous formaldehyde solution and subsequently embedding the stabilized samples for histological tissue studies. However, such a stabilization is in most cases unsuitable for the use of molecular-biological methods since the nucleic acids are only very insufficiently stabilized, which only makes possible a qualitative detection, at best, of the nucleic acids or nucleic acid fragments present, but not a quantitative detection. Moreover, the stabilization with crosslinking stabilizers such as aqueous formaldehyde solution leads to a reduced extractability of the nucleic acids or proteins from the tissues. Also, aqueous formaldehyde solution is not acceptable for toxicological reasons.
Stabilizing reagents such as, for example, the cationic detergents described in U.S. Pat. Nos. 5,010,184, 5,300,545, WO-A-02/00599 and WO-A-02/00600, which, in turn, give very good qualitative detection of the nucleic acids, are only suitable for samples which comprise single cells, or only one cell layer. To stabilize nucleic acids in compact pieces of tissue, however, such stabilizing reagents are not sufficient.
Moreover, those reagents and methods with which nucleic acids can be stabilized for the purposes of qualitative detection are, as a rule, not suitable for the simultaneous stabilization of proteins. Moreover, samples stabilized in this manner cannot be used for histological study since the stabilizer preserves for example the nucleic acids, but not the cell or tissue structures. Yet other stabilizing reagents which comprise, for example, highly concentrated ammonium sulfate (see, for example, U.S. Pat. No. 6,204,375) are well suited to the stabilization of nucleic acids in different tissues. However, they are largely unsuitable for use in the stabilization of cell-containing or cell-free body fluids such as, for example, blood, serum or plasma, and also have not as good stabilizing properties in some types of tissue, such as, for example, fatty tissue.
All the above shows that it is particularly difficult simultaneously to stabilize RNA, DNA and proteins in tissue samples and histologically to preserve the tissue samples. Moreover, work carried out on cells or other biological samples cannot necessarily be applied to compact tissue. In comparison with other biological samples, the stabilization of nucleic acids in compact tissue samples involves one particular difficulty. Tissues are composed of several layers and are heterogeneous with regard to their composition, their constituents and their structure. To stabilize nucleic acids in compact tissue samples, the stabilizing reagent must act not only on the cell surface, or within one cell layer, but also deep inside the multi-layer sample material. Moreover, one frequently has to address, within one and the same biological sample, very different types of tissue and/or cells, which differ for example with regard to their cell structure, the membrane construction, the compartmentalizations and the biomolecules, for example with regard to the proteins, the carbohydrates and/or the fat content.
One form of stabilizing tissue samples, including all constituents, which is known in the prior art and used very frequently is to freeze or deep-freeze the samples. Here, the sample is frozen in its natural environment in liquid nitrogen at below −80° C., immediately after having been taken. The sample treated thus can then be stored virtually indefinitely at approximately −70° C., without any changes in its integrity taking place. However, all such methods require very complicated logistic requirements since defrosting of the samples during transport, storage or during a wide range of purposes and utilizations must be prevented. Besides the additional costs for specific sample receptacles and for the permanent cooling of the samples, the use of liquid nitrogen is not only very complicated, but also can only be carried out with specific precautionary measures.
Moreover, a subsequent analysis of the frozen sample material, in particular individual components of the sample, is usually a very difficult endeavor. For example, defrosting, or incipient defrosting, of the sample during storage, transport or methoding leads to the degradation of, in particular, the RNA. This means that samples which have been subjected to defrosting, or incipient defrosting, no longer give reproducible results. In addition, it is precisely tissue pieces in the frozen state which are very difficult to method, for example divide, manually, or only with complex technical equipment.
Solutions referred to as transition solutions have also been described for lessening the disadvantages of methoding frozen samples, in particular for isolating RNA. Here, the frozen tissue is first transferred into a solutions precooled to −70° C. to −80° C., where it is stored for several hours (at least 16 hours) at approximately −20° C. Thereafter, the sample which is impregnated with the transition solution may be warmed to working temperatures of from −4° C. up to room temperature, for a brief period only, for example no longer than is necessary for dividing the sample, without any changes taking place in the nucleic acid status of the sample. However, further analyses, and storage of the sample, in particular at room temperature, are not possible. Such transition solutions which are known for example from WO-A-2004/72270 consist predominantly of monohydric alcohols.
The disadvantage of the samples treated with customary transition solutions is that they only remain stable at room temperature over a very short period, which means that the methoding time is only very limited and very readily exceeded in particular when methoding a large number of samples, in particular when cutting and chopping procedures are involved. Moreover, the transition is only very slow, whereby no direct experiments may follow, and waiting times of in most cases one day result. Equally, transport of the samples treated thus is not possible at room temperature without the sample being damaged, since not only must the transition take place at temperatures of ≦−20° C., but this must be followed by stable storage of the sample. Also, the transport of the sample is only possible at ≦−20° C., which requires the use of cooling means, for example dry ice, during transport. Furthermore, it must be taken into consideration that the monohydric alcohols employed in WO-A-2004/72270, such as, for example, methanol, ethanol or isopropanol, are readily flammable, volatile or toxic, and that, accordingly, certain safety precautions must be in place when using them.
While the use of the traditional transition solutions leads to improvements in sample methoding, such as, for example, chopping or cutting to size, they neither reduce the equipment requirements (since the solution for transition must be precooled at −70 to −80° C., and therefore still requires a suitable cooling device), nor is it possible to stabilize the transition-solution-treated samples at room temperature over a prolonged period.
The present invention was based on the object of overcoming the disadvantages of the prior art.